The Rotor-Gene SYBR Green RT-PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly specific quantification of RNA targets with real-time one-step RT-PCR using SYBR Green I detection. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

When the Rotor-Gene SYBR Green RT-PCR Kit is used together with QuantiTect Primer Assays, highly sensitive quantification of specific PCR products is achieved without the need for optimization (see figures "Specific detection without the need for optimization").

Principle

The Rotor-Gene SYBR Green RT-PCR Kit enables reliable real-time RT-PCR quantification on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. Real-time one-step RT-PCR is carried out, which means that RNA is used as template in a reaction where reverse transcription and PCR take place sequentially in the same reaction vessel. Since it is not necessary to transfer the finished RT reaction to another tube for PCR, the real-time RT-PCR procedure is streamlined, making high-throughput analysis possible.

The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that considerably shortens cycler run times (see figure "Fast primer annealing").

Components of 2x Rotor-Gene SYBR Green RT-PCR Kit*

Component

Features

Benefits

HotStarTaq Plus DNA Polymerase

5 min activation at 95ºC

Set up of qPCR reactions at room temperature

Rotor-Gene SYBR Green RT-PCR Buffer

Balanced combination of NH4+ and K+ ions

Specific primer annealing ensures reliable qPCR results

Unique Q-Bond additive

Faster PCR run times enable faster results and more reactions per day

SYBR Green I dye

Yields a strong fluorescent signal upon binding double-stranded DNA

Highly sensitive quantification

Rotor-Gene RT Mix

Special blend of reverse transcriptases with a high affinity for RNA

RNA can be transcribed in just 10 minutes, even through complex secondary structures

* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).

Procedure

A ready-to-use master mix eliminates the need for optimization of reaction and cycling conditions. Simply add template RNA, primers, and the supplied reverse transcriptase mix to the master mix and program the cycler. Instructions are provided in the detailed handbook supplied with the kit.

For gene expression analysis using real-time one-step RT-PCR, the combination of the Rotor-Gene SYBR Green RT-PCR Kit with QuantiTect Primer Assays and the Rotor-Gene Q provides a complete, ready-to-run solution. QuantiTect Primer Assays are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.

Applications

The Rotor-Gene SYBR Green RT-PCR Kit provides rapid real-time quantification of RNA targets on the Rotor-Gene Q. The kits are also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000.

Feature

Specifications

Applications

Real-time quantification of RNA targets

Description

For ultrafast quantitative real-time one-step RT-PCR using SYBR Green I